Evaluation of the anti-dandruff activity – Aim, Materials & Methods – GREENQUAT®BT

AIM

The aim of the test is to evaluate the efficacy of the tested cosmetic preservative to reduce the microbial load of a strain following the Minimun Inhibitory Concentration and Minimum Bactericidal protocols using appropriate microbiological techniques.

MATERIALS AND METHODS

Microrganisms used
The microorganisms used to evaluate the antimicrobial activity of the cosmetic preservative have been previously agreed with the customer and are listed below:
✓ GRAM POSITIVE COCCI:
Propionibacterium acnes (ATCC 11827);
✓ YEASTS AND MOULDS:
Malassezia furfur (ATCC 14521).

Growth media
The growth medium used for the MIC analisys is:
– TSB (Tryptone Soy Broth, Condalab, COD: 1224).
– BHI (Brain Heart Infusion Broth, Condalab, COD: 1400
The growth media used for the preparation of the MBC subcultures are:
– Blood Agar Base N°2 (Condalab, COD: 0912);
– SDA (Sabouraud Dextrose Agar, Condalab, COD: 1024) added with 0.5% Tween 80 (Sigma Aldrich, P1754-500ML).

Determination of microbial load
The ability of the tested cosmetic preservative to reduce the microbial load of the strains was evaluated by the Mininum Inhibitory Concentration (MIC) in a 96-wells plates.

Preparation of the sample and inoculation
Serial dilutions of the tested preservative were prepared on a 96-wells plates (11% – 10% – 9% – 8% – 6% – 5% – 4% – 3% – 2%) diluting in specific medium broth for the selected strains.
Samples (180 μl) of each dilutions or medium broth (for the inoculum control) were put on 96-wells plates; then 20 μl of the microbial strain were added (final concentration 106 CFU/ml). Plates were incubated for 24 h at 37°C in anaerobiosis growth condition for P. acnes and 24 h at 30°C for M. furfur, respectively.

Evaluation of bactericidal activity
Once the MIC was visually determined in a 96-well plate, 10 μl of the control, the MIC and following concentrations (increasing and decreasing with respect to the MIC) were plated in duplicate, in particular Blood Agar Base N°2 plates incubated for 72 h at 37 °C for anaerobic bacteria, SDA + Tween 80 plates incubated for 72/120 h at 30 °C for M. furfur. After incubation time, the colonies on the plate were enumerated in order to calculate the reduction of the microbial load obtained from each of the tested concentrations compared to the control.
The same protocol was followed for all selected strains.